The cytochrome P450 system plays an important role in metabolism of endogenous compounds and xenobiotics. This system consists of cytochrome P450, NADPH:cytochrome P450 oxidoreductase (CPR), cytochrome b5 and NADH:cytochrome b5 reductase (CYB5R3). Explanation of protein-protein interactions among these reaction partners is essential for understanding the function of the entire system. Covalent cross-linking is a favorable method for studying these interactions. In this work a photo-activatable analogue of amino acid L-methionine (L-photo-methionine) was used as a cross-linking agent. This work is focused on the organic synthesis of L-photo-methionine, expression and isolation of CPR and CYB5R3 as photoactivable proteins containing incorporated L-photo-methionine. Auxotrophic strain of E.coli B834 (DE3) and minimal media were used for the expression. CYB5R3 with incorporated L-photo-methionine was successfully expressed and isolated. The extent of L-photo-methionine incorporation was verified by mass spectrometry. Furthermore, the photo-initiated cross-linking of CYB5R3 with cytochrome b5 was tested. Key words: photolabile amino acid, protein expression, synthesis
| Identifer | oai:union.ndltd.org:nusl.cz/oai:invenio.nusl.cz:353847 |
| Date | January 2015 |
| Creators | Dědič, Jan |
| Contributors | Hodek, Petr, Novák, Petr |
| Source Sets | Czech ETDs |
| Language | Czech |
| Detected Language | English |
| Type | info:eu-repo/semantics/masterThesis |
| Rights | info:eu-repo/semantics/restrictedAccess |
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